Molecular strategies to enhance induced pluripotent stem cell reprogramming in buffalo (Bubalus bubalis)

1 Introduction

Induced pluripotent stem cells (iPSCs) are somatic cells reprogrammed to a pluripotent state, similar to embryonic stem cells (ESCs) (Takahashi and Yamanaka, 2006; Takahashi et al., 2007). Once obtained and characterized, these cells can differentiate in vitro into all embryonic tissues (mesoderm, endoderm, and ectoderm), including primordial germ cells (PGCs), which can subsequently give rise to gametes (oocytes and sperm – in vitro gametogenesis). This capability enables possibilities for their application in reproductive biotechnology (Pessôa et al., 2019).

iPSCs have been produced from different mammalian species, such as primates (Geuder et al., 2021; Yoshimatsu et al., 2021b), mice (Li et al., 2011; Hou et al., 2013), porcine (Ezashi et al., 2009; Pieri et al., 2021; Recchia et al., 2022), cattle (Su et al., 2021; Recchia et al., 2025), horse (Pessoa et al., 2019; Bressan et al., 2020) and buffalo (Deng et al., 2012; Thangavel et al., 2016). However, the full elucidation of the reprogramming process in Non-model animals iPSCs remains unclear, and further studies are necessary. For example, the type of somatic cell being reprogrammed can influence the success of iPSCs generation, with the majority of cell types used being fetal or adult fibroblasts. Other factors that may influence cellular reprogramming and are being investigated in animal models include transcript combinations and vector systems (integrative and non-integrative) (Bressan et al., 2020; Yoshimatsu et al., 2021a). Therefore, the successful production and characterization of iPSCs in these farm animals represent the first step toward the in vitro production of gametes and embryos using these technologies.

Buffaloes (Bubalus bubalis) are one of the key species in livestock production and are particularly targeted for the development of reproductive biotechnologies (Baruselli et al., 2023). Their significant role in this sector drives the advancement of technologies aimed at improving herd efficiency without increasing environmental impacts. In this context, reproductive biotechnologies are important, as they enhance animal reproduction efficiently, while minimizing the environmental burden associated with livestock farming (Hufana-Duran and Duran, 2020). However, despite the species’ high potential, several challenges still limit the efficient application of assisted reproductive technology in buffaloes.

Reproductive biotechnologies, such as artificial insemination, in vitro embryo production, and cloning enhance the efficiency of buffalo herds by ensuring improved reproductive performance, thereby promoting the dissemination and stabilization superior genetic traits within the herds at an accelerated pace. Such technologies are becoming increasingly prevalent in the livestock sector, including in buffalo production (Srirattana et al., 2022).

However, the buffaloes present specific characteristics that pose challenges during the application of reproductive biotechnologies, ultimately reducing their efficiency (Mitio Ohashi et al., 2017). Issues such as inadequate animal management and health, as well as reproductive traits of the species (such as reduced follicular population compared to bovine and diminished or absent estrous signs), can directly influence the outcomes of reproductive biotechnologies, resulting in low gamete recovery rates and decreased quantity and quality of embryos produced (Marin et al., 2019).

Regarding buffalo iPSC generation, limited information is available on the species-specific challenges associated with reprogramming and the maintenance of iPSCs. To date, only a few studies have addressed the generation of buffalo iPSCs derived from fetal cell lines (Deng et al., 2012; Thangavel et al., 2016; Mahapatra et al., 2017) which are less differentiated than adult somatic cells. Buffaloes have been reported to display molecular and metabolic features that may distinguish them from other species in which iPSC production technologies are already well established.

These peculiarities can pose challenges during the reprogramming process, as existing protocols are primarily based on species with well-documented reprogramming processes, such as human and mice. Therefore, understanding these specific characteristics may assist in an optimized protocol for cellular reprogramming in this species, ensuring improved efficiency in the production of iPSCs.

2 Principles and challenges of buffalo cellular reprogramming

iPSCs represent a significant milestone in the field of stem cells and cell therapy by confirming that specialized cells can be reprogrammed to an undifferentiated state, in addition to providing several advantages such as easy accessibility (since any donor somatic cell can be reprogrammed), the possibility of autologous use in therapy, a known animal genetic background, a high expansion rate, and the potential to obtain cell from the three germ layers (Gorecka et al., 2019; de Castro et al., 2024).

iPSCs are developed through the epigenetic reprogramming of somatic cells induced by the overexpression of the transcription factors OCT3/4, SOX2, c-MYC, and KLF4 (the OSKM factors, which are associated with the maintenance of pluripotency, proliferation, and cell cycle control), introduced into the cells using integrative or non-integrative vectors (Bao et al., 2024). Following transfection, the cells begin to exhibit morphological and physiological characteristics similar to those of ESCs (embryonic stem cells), such as unlimited proliferative and differentiation potential, growth in colonies with well-delimited borders, a gene expression profile similar to that of ESCs, the ability to generate teratomas, and the capacity to be derived from differentiated somatic cell types (Takahashi and Yamanaka, 2006).

However, reprogramming to pluripotency involves a series of cellular events that include nearly complete epigenetic remodeling, as well as metabolic modulation, leading to significant alterations in the activity of key cellular pathways, particularly those associated with cell proliferation and differentiation, which are hallmark characteristics of embryonic cells (Ishida et al., 2020).

Once the iPSCs are generated, it has been reported that they can hold different levels of pluripotency: the naïve state, which corresponds to the inner cell mass (ICM) of the blastocyst, and the primed state, which is similar to the epiblast just before implantation, when the ICM has differentiated into the hypoblast and epiblast. Each pluripotent state of pluripotency exhibits distinct gene expression profiles, epigenetic signatures, and specific morphological characteristics (Messmer et al., 2019).

Over the past few years, the technology for producing iPSCs has been extended to various other species. During the process of reprogramming, optimized protocols have been shown to be necessary for different species. These optimizations include different combinations of reprogramming factors (in addition to the commonly used OSKM factors) and different reprogramming methodologies, using either integrative vectors (e.g. lentiviral), in which the transcription factors are integrated into the cell genome, or non-integrative (e.g. episomal), where no exogenous DNA is integrated into the cell genome (Miskey et al., 2022; Pessôa et al., 2019; Yoshimatsu et al., 2021a).

One of the central molecules involved in maintaining pluripotency in both ESCs and iPSCs is basic fibroblast growth factor (bFGF), which sustains high expression levels of key pluripotency factors such as OCT4, SOX2, and NANOG, thereby preventing differentiation. Mechanistically, bFGF signals through fibroblast growth factor receptors (FGFRs) to activate multiple downstream pathways, among which the MAPK cascade represents the major axis regulating pluripotency (Haghighi et al., 2018).

Likewise, the use of small molecules capable of modulating key signaling pathways associated with the pluripotent status of cells has also been widely explored, particularly those involved in pathways such as WNT/β-catenin, TGF-β/Activin/Nodal, FGF/ERK (MAPK), and PI3K/AKT signaling, which play central roles in the regulation and maintenance of pluripotency (Kim et al., 2020).

In several domestic animal species, a higher reprogramming efficiency, or even successful reprogramming exclusively through the use of integrative methodologies has been already reported. However, non-integrative methodologies are particularly attractive due to the possibility of generating iPSCs without transgenesis, thereby avoiding the production of genetically modified animals or products, as well as potential instabilities in the silencing of exogenous transcripts (Bressan et al., 2020; Machado et al., 2021; Pessôa et al., 2019).

Furthermore, the generation of iPSCs through non-integrative approaches has been successfully reported in association with the modulation of pathways such as WNT/β-catenin (Zhang et al., 2014), TGF-β/SMAD (Fan et al., 2024), and JAK/STAT3 (Wang et al., 2018; Recchia et al., 2025; Yoshimatsu et al., 2021a; Wang et al., 2018). The modulation of these pathways also occurs in a species-specific manner; therefore, their role remains incompletely understood in domestic and wild species (Su et al., 2020).

In buffalo, the available evidence remains limited, but the published studies indicate that somatic cell reprogramming is technically feasible using both integrative and non-viral approaches (Tables 1, 2).

Table 1. Buffalo studies on cellular reprogramming and iPSC generation, including the use of VPA and egg/oocyte extracts.

Table 2. Culture media for pluripotent cells in bovine and buffalo contain various essential components that modulate key signaling pathways involved in cellular and metabolic processes critical for embryonic development, such as cell proliferation and differentiation.

The first buffalo-specific report demonstrating robust iPSC generation reprogrammed buffalo fetal fibroblasts using retroviral delivery of buffalo-derived OCT4, SOX2, KLF4, and c-MYC. The resulting colonies displayed typical pluripotent stem cell morphology, alkaline phosphatase (AP) activity, normal karyotype, expression of pluripotency-associated markers, embryoid body formation, and in vivo teratoma formation(REF – tava aqui só inc. o texto).Importantly, this study also provided the strongest functional validation reported so far in buffalo, as the authors demonstrated hypomethylation of the OCT4 and NANOG promoters, development of reconstructed embryos to the blastocyst stage after nuclear transfer, and an approximately threefold increase in reprogramming efficiency following p53 inhibition (Deng et al., 2012).

Subsequent studies indicated that buffalo fetal fibroblasts can also undergo reprogramming through lentiviral delivery of mouse OSKM factors, yielding AP–positive colonies with normal karyotype, expression of canonical pluripotency markers, in vitro trilineage differentiation, and sustained proliferation for more than 20 passages (Thangavel et al., 2016).

Later studies moved beyond proof-of-concept and focused on protocol optimization and alternative delivery strategies. A non-viral piggyBac system carrying six reprogramming factors—OCT4, SOX2, KLF4, c-MYC, NANOG, and Lin28—was subsequently applied to buffalo fetal fibroblasts, generating putative iPSC colonies under feeder-free conditions. Colony formation was observed after 12–14 days, and the resulting cell lines exhibited in vitro differentiation into all three germ layers.

This strategy aligns conceptually with transposon-based reprogramming approaches reported in other livestock species, where removable non-viral cassettes have been investigated as alternatives to retroviral and lentiviral vectors to mitigate concerns associated with insertional mutagenesis (Kumar et al., 2019).

In parallel, mechanistic work by Luo et al. (2022) showed that overexpression of buffalo OCT4, c-MYC, KLF4, and SOX2 activates signaling pathways associated with early reprogramming, including LIF, activin, BMP4, SMAD1/5/9, and Wnt, thereby providing a molecular framework for future optimization using small molecules and pathway modulation (Luo et al., 2022).

Additional buffalo studies have indicated that reprogramming efficiency is highly dependent on epigenetic modulation and culture conditions. Treatment with valproic acid has been shown to enhance reprogramming in buffalo fetal fibroblasts transduced with mouse OSKM, with 1.5 mM identified as the optimal concentration. Under these conditions, the number of GFP-positive colonies increased by approximately 2.5-fold, while ESC-like morphology, normal karyotype, and trilineage differentiation capacity were maintained (Mahapatra et al., 2017).

Alternative noncanonical approaches have also been explored. Exposure of buffalo fibroblasts to chicken egg extract has been shown to induce AP–positive colonies and to increase the expression of pluripotency-associated genes, including OCT4, SOX2, NANOG, and FOXD3, in a dose-dependent manner (Mahapatra and Bag, 2014). In a related line of investigation, buffalo oocyte extracts were found to elevate OCT4 and NANOG expression while reducing HDAC1, DNMT1, and DNMT3A levels, consistent with partial epigenetic resetting of donor fibroblasts (Sadeesh et al., 2017). Taken together, these findings indicate that buffalo cell reprogramming can be achieved through multiple routes, yet they also underscore the absence of a stable and fully validated induction system, particularly regarding long-term maintenance, transgene silencing, and precise characterization of the resulting pluripotent state.

Within the broader context of pluripotency research in buffalo, efforts to establish embryonic stem cells (ESCs) lines have centered on deriving ESCs from the ICM of in vitro-produced preimplantation embryos.

Derivation efficiency of pluripotent colonies has been shown to vary substantially according to the developmental stage of the donor embryo and the strategy used for ICM isolation. Hatched blastocysts consistently produce higher frequencies of primary colony formation compared with early or expanded blastocysts, whereas morula-derived blastomeres do not generate stable primary colonies. Mechanical isolation of the ICM yields more robust outcomes than enzymatic dissociation, which often triggers premature differentiation and restricts colony maintenance to only a few passages. The resulting colonies typically exhibit AP activity, maintain a normal karyotype, express OCT4, and generate embryoid bodies containing neuronal-like and epithelial-like derivatives (Verma et al., 2007). Huang et al. (2010) confirmed this stage- and method-dependent pattern in an independent cohort of IVF-derived embryos.

An ESCs line has been generated from a buffalo parthenogenetic embryo, demonstrating expression of key pluripotency-associated markers and the capacity for multilineage differentiation under in vitro conditions. The line also formed teratomas when introduced into immunodeficient mice, providing early in vivo confirmation of pluripotent characteristics in buffalo ESCs (Sritanaudomchai et al., 2007).

Subsequent studies established ES-like lines from additional embryonic sources, including cloned and somatic cell nuclear transfer (SCNT)-derived blastocysts. ES-like cells obtained from cloned blastocysts exhibited dome-shaped colony morphology and expressed a pluripotency marker profile comparable to that observed in IVF-derived lines. These cells also showed spontaneous differentiation, predominantly toward endodermal and epithelial lineages, further supporting the developmental competence of ES-like cells derived from SCNT embryos (George et al., 2011).

ES-like lines have also been generated and compared from IVF, parthenogenetic, and cloned buffalo embryos within a single experimental framework. Primary colony formation occurred in approximately 45–55% of isolated inner cell masses across all embryonic origins, and the resulting lines consistently exhibited AP activity, normal karyotype, and a broad pluripotency marker profile, including OCT4, NANOG, SOX2, SSEA-1/4, TRA-1-60/81, REX1, CD90, STAT3, and telomerase. These lines demonstrated trilineage differentiation, confirming their functional pluripotency (Shah et al., 2015).

A key limitation of earlier studies was the inability to maintain undifferentiated cells beyond 8–10 passages. Sharma et al. (2011) addressed this by culturing mechanically isolated ICMs on buffalo fetal fibroblast feeders in KO-DMEM supplemented with Knockout Serum Replacement (KSR), FGF-2, and LIF, maintaining three ES-like lines for 85–135 passages over 17–27 months. FGFR inhibition increased differentiation, and FGF-responsive signaling intermediates (ERK1, K-RAS, E-RAS, SHP-2) were detected in undifferentiated cells, implicating FGF signaling in pluripotency maintenance (Sharma et al., 2011).

These findings indicate that ESC derivation in buffalo depends on embryo availability, precise ICM handling, and optimized culture conditions, whereas iPSC generation relies instead on forced reprogramming via vector-mediated delivery of exogenous factors. Buffalo ESC lines have nonetheless been captured from multiple embryonic origins and, under optimized conditions, maintained more extensively than most reported iPSC lines. The ESC literature thus provides a benchmark of pluripotency-associated phenotypes and culture dependencies against which reprogrammed buffalo cells can be evaluated, and remains central to defining stable, functionally credible pluripotency in this species.

These factors may hinder the direct application of protocols that have already been established for reprogramming in other model species, including humans, mice, and even bovines (Scarfone et al., 2020). Several approaches that can be employed to enhance reprogramming efficiency in buffalo cells, including strategies that modulate cellular signaling and metabolic pathways, thereby improving the acquisition of pluripotency and sustaining the expression of endogenous pluripotency genes over extended in vitro passages.

3 Histone deacetylases inhibitors in reprogramming in buffaloes

The process of reprogramming to pluripotency essentially involves a global remodeling of gene expression in cells, systematically and controllably altering gene expression patterns by silencing those related to somatic tissue functions and reactivating genes associated with proliferation and the maintenance of the undifferentiated status of the cells (Basu and Tiwari, 2021). This process of chromatin remodeling is carried out through chemical modifications to DNA and histones, such as methylation, acetylation, and phosphorylation, which are mediated by enzymes that control these modifications (Millán-Zambrano et al., 2022).

The activation or inhibition of these enzymes has a significant impact on reprogramming to pluripotency (Brix et al., 2015). One example of such enzymes is histone deacetylases (HDACs), which are responsible for removing acetyl groups from histones, thereby increasing their affinity for the DNA double helix and hindering the transcription machinery’s access to genetic information (Falkenberg and Johnstone, 2014). The use of histone deacetylase inhibitors (HDACi) during culture is associated with increased reprogramming efficiency, enhancing colony formation and improving their quality (including morphology and the expression of endogenous pluripotency genes) across different species (Basu and Tiwari, 2021) (Figure 1).

The initiation of somatic cell reprogramming is strongly dependent on the pioneer transcription factor OCT4, which can bind to chromatin regions that are normally inaccessible in differentiated cells and promote increased chromatin accessibility at loci associated with pluripotency. This early chromatin remodeling facilitates the recruitment of additional transcription factors such as SOX2 and KLF4 and enables the activation of the pluripotency transcriptional network. In this context, the use of histone deacetylase inhibitors during reprogramming further facilitates this process by increasing histone acetylation levels, thereby promoting chromatin relaxation and enhancing the accessibility of regulatory regions to the reprogramming factors (Li et al., 2021).

Several small molecules have been used as HDACis to enhance reprogramming efficiency, including valproic acid (VPA), trichostatin A (TSA), sodium butyrate (NaB), suberoylanilide hydroxamic acid (SAHA), among others (Kretsovali et al., 2012).

In reprogrammed human bone marrow cells, treatment with VPA improved cell proliferation in the early post-reprogramming stages, primarily by downregulating the activated p16/p21 pathway, thus reducing cellular senescence induced by lentiviral transduction. Furthermore, VPA was responsible for decreasing the activation of other senescence-related pathways, such as Caveolin-1, APO-J, and OX-1. It also promoted the activation of the anti-apoptotic BCL2 signaling pathway and alleviated cell cycle arrest in senescent cells at the G2/M phase (Chen et al., 2016).

In humans, the application of TSA has been shown to enhance the efficiency of gene editing in iPSCs using the CRISPR-Cas9 system. TSA facilitates increased chromatin decondensation, thereby improving gene editing precision and efficiency while maintaining genomic integrity. Furthermore, the use of TSA supports the preservation of cellular pluripotency, with no observable increase in off-target effects (Molugu et al., 2023).

In porcine, the use of three histone deacetylase inhibitors (VPA, NaB, and SAHA, along with ascorbic acid) was able to sustain the endogenous expression of pluripotency genes in iPSCs-like cells, including those associated with the early stages of embryonic development (the naïve state), for more than 60 passages without the use of feeder cell layers (Petkov et al., 2016). In bovines, the pre-treatment of donor cells with VPA for cloning purposes increased the reprogramming efficiency of these cells, resulting in a higher blastocyst formation rate compared to the untreated group. Additionally, the embryos produced showed reduced methylation at lysine 9 of histone 3 (H3K9me3), demonstrating the role of VPA in the epigenetic reprogramming of the donor cells (Selokar et al., 2013).

In buffalo, different concentrations of VPA were tested during the reprogramming of fetal fibroblasts using lentiviral vectors carrying mouse OSKM factors, and a concentration of 1.5 mM VPA yielded the most favorable results. In this group, a significant increase in the number and size of the colonies was observed. These colonies demonstrated chromosomal stability across passages, expressed pluripotency marker genes, and tested positive for AP activity as well as embryonic stem cell markers, including OCT4, NANOG, SSEA1, TRA-1-60, and TRA-1-81, compared with the untreated group (Mahapatra et al., 2017). These results underscore VPA’s role in enhancing the efficiency of epigenetic reprogramming in this species.

As demonstrated by the use of histone deacetylase inhibitors during reprogramming in several species, the underlying molecular mechanisms are primarily associated with chromatin relaxation resulting from increased histone acetylation. This process enhances chromatin accessibility, facilitating the activation of pluripotency-related genes and promoting the removal of residual somatic epigenetic marks from reprogrammed cells. In addition, these compounds may act synergistically with signaling pathways involved in reducing cellular senescence and promoting cell proliferation, such as the p16/p21 pathway and the TGF-β and MEK–ERK signaling pathways.

Furthermore, metabolic modulation holds promise as a strategy, particularly under hypoxic conditions, as well as through the activation of key developmental pathways, such as the WNT signaling pathway, which may further enhance reprogramming efficiency. These alternative methods expand the potential for optimizing the generation of high-quality iPSCs across different species, including buffaloes.

4 Reprogramming in low oxygen tension (hypoxia)

Hypoxia is characterized by a series of cellular responses, including changes in gene expression patterns and metabolic modulation, in response to low oxygen concentrations. This response is activated and regulated by the action of hypoxia-inducible factors (HIF), a family of transcription factors that modulate cellular behavior by altering signaling pathways and metabolism in response to reduced oxygen availability (Malkov et al., 2021) (Figure 2).

This response to low oxygen environments (<5%) is closely associated with stem cells, both in adult tissues and embryos, leading to several changes that enhance their proliferation, self-renewal, and differentiation potential. These changes include reduced damage to cellular structures, particularly DNA, due to the lower production of reactive oxygen species, as well as metabolic modulation, such as the activation of anaerobic glycolysis, among others (Hawkins et al., 2013).

In ESCs, this metabolic transition from oxidative phosphorylation to glycolysis is accompanied by increased expression of enzymes that catalyze key steps of the glycolytic pathway, such as hexokinase (HK), which catalyzes the phosphorylation of glucose to generate glucose-6-phosphate (G6P), and pyruvate kinase (PK), which catalyzes the conversion of phosphoenolpyruvate into pyruvate. Moreover, key pluripotency factors involved in cellular reprogramming, such as OCT4, directly regulate the expression of enzymes associated with the glycolytic pathway, thereby influencing the metabolic state of cells undergoing reprogramming (Ishida et al., 2020).

Additionally, HIFs also play a role in the epigenetic modulation of cells under hypoxic conditions, including DNA methylation and histone acetylation. This upregulation of the glycolytic pathway in pluripotent cells increases the availability of metabolic intermediates such as acetyl-CoA, α-ketoglutarate, and NAD⁺, which act as regulators of enzymes including histone deacetylases and histone acetyltransferases. These enzymes are responsible for modulating chromatin structure by maintaining appropriate levels of histone acetylation and thereby enabling the epigenetic remodeling required for successful cellular reprogramming (Garone et al., 2024).

They also alter the expression of non-coding RNAs, which can modulate the expression of key genes in response to the stress induced by low oxygen concentrations (Choudhry and Harris, 2018). One of the post-transcriptional regulatory mechanisms influenced by HIF is associated with the increased expression of the miR-302 cluster, which positively regulates NANOG expression in cells undergoing reprogramming by inhibiting the epigenetic repressor MBD2. Furthermore, the miR-302 cluster appears to be involved in the regulation of cell cycle progression and in the induction of the mesenchymal-to-epithelial transition (MET) during cellular reprogramming (Ferreira et al., 2018).

Exposure of differentiated human pluripotent cells (ESCs or iPSCs) to hypoxia has been shown to reactivate the OCT4 promoter, leading these cells to regain pluripotent characteristics, including their characteristic morphology, unlimited self-renewal capacity, membrane markers, and pluripotency-associated gene expression profiles. This process effectively reversed their differentiated status, returning them to a pluripotent state (Mathieu et al., 2013).

Another important characteristic of cells subjected to hypoxic conditions is the modulation of their metabolism, altering cellular behavior in terms of metabolic energy production and the synthesis of other biomolecules. This metabolic shift is commonly known as anaerobic glycolysis or the Warburg effect. Under normoxic conditions, glucose molecules used for ATP production through the glycolytic pathway and subsequent oxidative phosphorylation are converted into pyruvate. However, in cells with high proliferative rates and under hypoxic conditions, such as cancer cells and ESCs, the pyruvate produced is preferentially converted into lactate, redirecting energy metabolism in these cells toward the biosynthesis of other biomolecules (Fukushi et al., 2022).

In cancer cells, this metabolic behavior is characteristic and crucial for maintaining tumor homeostasis, allowing the reprogramming of the cellular population and adjusting ATP concentrations. This ensures the accumulation of glycolytic intermediates that can be used as precursors for the biosynthesis of cellular components, in addition to the accumulation of lactate, which contributes to the modulation of the anti-tumor immune response (Vaupel and Multhoff, 2021).

Regarding buffalo ESCs, proteomic analyses have demonstrated that embryos produced in vitro under hypoxic conditions, in addition to exhibiting better morphological quality and higher zona pellucida hatching rates, also showed several differentially expressed proteins related to glycolysis, fatty acid degradation, inositol phosphate metabolism, and terpenoid backbone synthesis. These findings are associated with the metabolic modulation that occurs during the Warburg effect (Shahzad et al., 2020).

For adult buffalo bone marrow mesenchymal stem cells (bBMSCs), there was a significant increase in colony formation and stress fiber synthesis of bBMSCs, and both the migration capacity and the normal karyotype rate of bBMSCs were significantly improved (Zhang et al., 2020). Hypoxia also enhanced the expression of pluripotency marker genes (OCT4, NANOG, and c-MYC) in mesenchymal stem cells derived from adipose tissue, which improved their efficiency in the reprogramming process into iPSCs, resulting in colonies with characteristics of naïve-like cells (Deng et al., 2019).

Therefore, a culture environment that provides low oxygen concentrations appears to be ideal for the reprogramming of buffalo cells towards pluripotency. This condition may assist not only in the epigenetic remodeling required for reprogramming, through both the expression of transcription factors and the activity of microRNAs, but also in the modulation of cellular metabolism and protection against oxidative damage.

5 Modulation of WNT pathway signaling in reprogramming

Another important molecular aspect of the reprogramming process towards pluripotency is related to the WNT/β-catenin signaling pathway, which has been increasingly associated with the maintenance of pluripotent characteristics in reprogrammed cells across various species (Figure 3).

There are 19 WNT protein-coding genes described in the mammalian genome, which are secreted as glycoproteins. The main signaling pathway involving WNT proteins is known as the canonical WNT pathway, or the WNT/β-catenin pathway. When a WNT protein binds to the Frizzled (FZD) membrane receptor, a series of intracellular changes occur within protein complexes, ultimately stabilizing the intracellular concentration of β-catenin. This accumulated β-catenin acts as a transcription factor that interacts with the DNA-binding factors of the TCF/LEF family (Azbazdar et al., 2021).

The WNT/β-catenin signaling pathway is associated with several important biological processes in mammals, ranging from axis formation during the early stages of embryonic development to the regulation of metabolic processes and cell proliferation. Dysregulation of this pathway is closely linked to the development of various pathologies, including multiple types of cancer in humans (Zhou et al., 2022).

In cattle, the importance of inhibiting the canonical WNT pathway for the derivation and establishment of pluripotent cell lines derived from embryos has already been demonstrated, using the inhibitor IWR-1, which was shown to block the activation of Signal Transducer and Activator of Transcription 3 (pSTAT3) in embryos, thereby preventing the differentiation of pSTAT3⁺ lineages (Xiao et al., 2021). The WNT pathway has also been shown to be crucial during the reprogramming of mouse fibroblasts to pluripotency, where the cytoplasmic accumulation of β-catenin interacted with the exogenous reprogramming factors OCT4, SOX2, and KLF4, thereby enhancing the expression of endogenous pluripotency-related genes (Zhang et al., 2014).

Several molecules have been employed both in the derivation and maintenance media of ESCs, as well as in the maintenance of iPSCs across various species, including buffalo, with the aim of modulating key pathways involved in pluripotency, differentiation, and cell proliferation mechanisms, such as the WNT/β-catenin pathway. Small molecules such as IWR-1, CHIR99021, XAV939 and 6-bromoindirubin-3′-oxime (BIO) specifically bind to proteins within the pathway’s complex, thereby activating or inhibiting it (Table 1).

In buffalo, activation of the canonical WNT pathway through inhibition of its antagonist GSK3, by adding BIO and CHIR99021 to the culture medium, improved the formation and proliferation rates of pluripotent stem cell colonies derived from blastocysts. Additionally, it enhanced the expression of genes associated with the pluripotency machinery, such as OCT4, SOX2, NANOG, and c-MYC, thereby ensuring the maintenance of the pluripotent state of these cells over multiple passages in culture (Deng et al., 2020). It has also been demonstrated that a culture medium that promotes WNT3A expression and contains exogenous fibroblast growth factor 2 (FGF2) and leukemia inhibitory factor (LIF) induces the proliferation of undifferentiated cells, thereby ensuring the maintenance of the pluripotent state (Zandi et al., 2014).

Due to the complexity of the components within the WNT pathway, its relationship with the upregulation of key genes associated with pluripotency is ultimately multifactorial, involving the participation of various elements at different stages of the signaling cascade. The β-catenin accumulated following WNT pathway activation can form complexes with other proteins, such as CREB-binding protein (CBP), which act as activators of the expression of genes like OCT4 and NANOG (Takao et al., 2007). Moreover, inhibition of GSK3, and consequently WNT activation, reduces repression of pluripotency-associated genes by TCF3 (a member of the TCF/LEF family), which is responsible for promoting the differentiation of pluripotent cells (Wray et al., 2011).

In addition to the complexity of the WNT pathway and the numerous molecules that can regulate its activity at various levels, such molecules are often small and may be nonspecific, binding to other proteins from different pathways and potentially exerting undesirable influences on cellular behavior. Therefore, culture medium formulations incorporating these molecules must be carefully tested, including their concentrations, to establish conditions that ensure the maintenance of pluripotency in both ESCs and iPSCs over multiple passages.

6 Conclusions

In conclusion, the studies compiled here indicate that although reprogramming protocols originally established in model species, including humans, mice, pigs, and cattle, have been applied with relative success in buffalo, reprogramming efficiency in this species remains consistently lower than that reported in reference systems (Table 3).

Table 3. Summary of HDACi, hypoxia, and WNT/β-catenin modulation in buffalo pluripotency research.

The generation of buffalo induced pluripotent stem cells using retroviral and lentiviral OSKM-based factors, as well as non-viral systems such as piggyBac, demonstrates the technical feasibility of cellular reprogramming in buffalo. However, it also highlights a strong reliance on exogenous factors and heterologous protocols, in the absence of a robust understanding of the species-specific regulatory mechanisms that govern pluripotency and cell fate transitions.

Although buffalo iPSCs and ESCs cells have been characterized by broadly accepted criteria, including dome-shaped colony morphology, AP activity, expression of core pluripotency markers, normal karyotype, and three-germ-layer differentiation potential, these parameters have more consistently confirmed pluripotent identity at the time of evaluation than long-term stability of the undifferentiated state.

This knowledge gap is particularly evident across the three major axes discussed in this review. HDAC inhibitors such as valproic acid have been evaluated only during fibroblast reprogramming, with no evidence yet regarding their role in the maintenance of established buffalo pluripotent lines. Likewise, hypoxia has been investigated mainly in buffalo mesenchymal stem cells and embryos, and its contribution to pluripotency maintenance in this species still depends largely on extrapolation from human and murine systems. Similarly, although WNT/β-catenin signaling has been implicated in both reprogramming induction and the maintenance of buffalo ESCs, direct comparative evidence with the canonical inhibitory mechanisms described in cattle remains limited.

Taken together, these discrepancies support the central premise of this review: interspecies variation in the expression and modulation of key signaling pathways associated with pluripotency, even between closely related species such as cattle and buffalo, constrains the direct transfer of heterologously optimized protocols. Progress in buffalo reprogramming will therefore depend on a species-specific characterization of the molecular networks that sustain and stabilize the undifferentiated state.

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Footnotes

Edited by: Francisco Javier Navas González, University of Cordoba, Spain

Reviewed by: Chenglei Tian, Helmholtz Center Munich, Germany

Yao Xiao, Shandong Academy of Agricultural Sciences, China